This invention relates to a test system and a procedure having an extended range of measurement for the determination of NAD(P)H or of substrates or enzymes which react with the formation or consumption of NAD(P)H.
In the majority of cases, the determination of clinical parameters is carried out using highly specific dehydrogenase reactions during the course of which NAD(P)H is indirectly or directly formed or consumed. The amount, absolute or per unit of time, of NAD(P)H reacted thereby is a measure of the concentration in a fluid of the substance to be investigated.
The dehydrogenase reaction has proved to be especially advantageous in respect of both stoichiometry and a low susceptibility to interference. On the other hand, it is a disadvantage that, due to the absorption properties of the coenzyme molecule, evaluation of the reaction for measurement is only possible using photometers with a UV measurement range; purely visual evaluations cannot be carried out. The latter are only made possible by coupling the actual reaction which forms NAD(P)H to a color reaction. A large number of procedures achieve this by direct transfer, or transfer using electron transfer agents, such as phenazine methosulphate or diaphorase, of the redox equivalents to a variety of redox indicators. The latter substances include, for example, cytochromes, complexed or chelated iron ions, dichlorophenol-indophenol, tetrazolium salts and the like. Coupling the reaction for measurement to a reaction sequence is also known (See, e.g., German Auslegeschrift No. 1,598,263, or European Patent Specification No. 54,146). The formation of a dyestuff takes place via a substance formed as an intermediate. A sequential course of reactions, in which each product from the first part reaction is the starting substance for the second and so on, is common to all these systems.
A procedure is described in German Patent Application No. P 32 11 167 in which a substance to be determined is reacted to give a variety of products which can be differentiated. Compared with the conventional tests, a wider range of measurement with the same accuracy of measurement is obtained. This is achieved by using several enzyme systems which, independently of one another, react with the same substance to be determined, one system being a NAD-dependent dehydrogenase and another being a NAD-independent dehydrogenase. A disadvantage of this procedure is that the NAD-independent enzymes necessary for a major proportion of the substances to be investigated in clinical diagnostics are not commercially available, in contrast to the corresponding NAD-dependent enzymes.